Jayshree Rajyaguru

X-ray micrography and imaging of Escherichia coli cell shape using laser plasma pulsed point x-ray sources.

High-resolution x-ray microscopy is a relatively new technique and is performed mostly at a few large synchrotron x-ray sources that use exposure times of seconds. We utilized a bench-top source of single-shot laser (ns) plasma to generate x-rays similar to synchrotron facilities. A 5 microlitres suspension of Escherichia coli ATCC 25922 in 0.9% phosphate buffered saline was placed on polymethylmethyacrylate coated photoresist, covered with a thin (100 nm) SiN window and positioned in a vacuum chamber close to the x-ray source. The emission spectrum was tuned for optimal absorption by carbon-rich material. Atomic force microscope scans provided a surface and topographical image of differential x-ray absorption corresponding to specimen properties. By using this technique we observed a distinct layer around whole cells, possibly representing the Gram-negative envelope, darker stained areas inside the cell corresponding to chromosomal DNA as seen by thin section electron microscopy, and dent(s) midway through one cell, and 1/3- and 2/3-lengths in another cell, possibly representing one or more division septa. This quick and high resolution with depth-of-field microscopy technique is unmatched to image live hydrated ultrastructure, and has much potential for application in the study of fragile biological specimens.

X-ray microscopy and imaging of Escherichia coli, LPS and DNA.

Ultrastructural examination by transmission and scanning electron microscopy involves a series of specialized preparation steps which may introduce artefacts in the micrographs. X‐ray microscopy can take instant images of speci‐mens but is mostly restricted to a few synchrotron X‐ray sources. We have utilized a bench‐top nanosecond laser‐plasma to produce a single‐shot source of nanosecond X‐rays tuned for maximum contrast with carbon‐rich material. To examine the ultrastructure by absorption profiles, we utilized a laser‐produced plasma generated by a single‐shot laser (1.06 μm wavelength, 5 × 1012 W cm−2 intensity) focused on to a silicon target as an X‐ray source for high‐resolution X‐ray microscopy. This approach eliminates the specimen preparation steps. Whole hydrated cells of Escherichia coli and purified preparations of lipopolysaccharide (LPS) and chromosomal DNA (cDNA) were streaked onto poly(methyl methacrylate) (PMMA)‐coated grids (resist). This resist was exposed to X‐rays under vacuum at a distance of 2.5 cm from the target disc. The silicon plasma produced by a 10‐ns burst of laser energy (at 20 J) radiates strong emission lines in the region of 300 eV. The X‐rays penetrate the sample and their absorption profile is transferred on to the resist where PMMA acts as a negative to generate an image. By atomic force microscopy imaging of this photoresist we have visualized layers around cells of E. coli, darker areas inside the cell probably corresponding to cDNA, and preliminary images of LPS and DNA molecules. This technique has resolution at the 100 Å level, produces images similar to the space‐filling models of macromolecules and may be of great value in the study of the ultrastructure of hydrated live biological specimens.

Ultrastructural imaging and molecular modeling of live bacteria using soft x-ray contact microscopy with nanoseconds laser-plasma radiation

X-ray images of the various live bacteria, such as Staphylococcus and Streptococcus, and micromolecule such as chromosomal DNA from Escherichis coli, and Lipopolysacchride from Burkholderia cepacia, are obtained with soft x-ray contact microscopy. A compact tabletop type glass laser system is used to produce x-rays from Al, Si, and Au targets. The PMMA photoresists are used to record x-ray images. An AFM (atomic force microscope) is used to reproduce the x-ray images from the developed photoresists. The performance of the 50nm spatial resolutions are achieved and images are able to be discussed on the biological view.

Fine surface structure of unfixed and hydrated macrophages observed by laser-plasma x-ray contact microscopy

A compact, high-resolution, laser-plasma, x-ray contact microscope using a table-top Nd:glass laser system has been developed and utilized for the analysis of the surface structure of live macrophages. Fine fluffy surface structures of murine peritoneal macrophages, which were live, hydrolyzed and not sliced and stained, were observed by the x-ray microscope followed by analysis using an atomic force microscopy. In order to compare with other techniques, a scanning electron microscopy (SEM) was utilized to observe the surface structure of the macrophages. The SEM offered a fine whole cell image of the same macrophages, which were fixed and dehydrated, but the surfaces were ruffled and different from that of x-ray images. A standard light microscope was also utilized to observe the shape of live whole macrophages. Light microscopy showed some fluffy surface structures of the macrophages, but the resolution was too low to observe the fine structures. Thus, the findings of fine fluffy surface structures of m...

Biological X-Ray Microscopy with a Compact Laser System

We describe progress being made in an x-ray imaging technology that provides high-resolution single frame x-ray images of in-vitro specimens captured in a time sufficiently short that radiation damage mechanisms to the structure are not recorded. Several different biology and medical research groups find this type of microscopy particularly well-suited to the detailed analysis of sub-cellular features, and to the study of live organisms subjected to various forms of external stimuli. This technology utilizes bright x-ray sources produced by compact pulse laser systems. The incorporation of advanced x-ray optical and electron-optical systems will lead to the development of a compact, real-time x-ray microscope, having a broad range of applications.

Nanosecond-resolved biological x-ray microscopy with a compact laser system

Nanosecond flash x-ray microscopy of living biological specimens is demonstrated with subcellular spatial resolution. Single shot images, produced by a compact laser- plasma x-ray source optimized for maximum image contrast, are captured before radiation processes can affect the specimen.